APOBEC3G (A3G) can restrict HIV by inducing catastrophic mutations in the viral genome. Critical to the consideration in the proposed studies, the HIV Vif protein binds to A3G and thereby induces proteasomal degradation of A3G. Despite much academic research on Vif, only a limited effort by industry and research institutions has gone toward identifying small molecule antagonists of Vif. OyaGen, Inc is the only commercial entity currently pursuing the identification of chemical scaffolds that specifically interact with if in meaningful ways for drug development. The significance of the proposed research is that Vif remains a new antiviral target whose clinical potential remains to be explored. An inherent limitation of prior anti-Vif drug discovery efforts has been the use of primary drug screens that have a mixed number of targets affecting the outcome of the screen. We propose an innovative drug discovery critical path that begins with a uniquely designed quenched FRET (FqRET) reporter assay as a stable cell line that selects specifically for antagonists of protein-protein interactions between Vif and A3G. The Specific Aims of the revised application will identify initia hits by high throughput screening of a large diversity set library to identify chemical scaffolds that have a concentration-dependent ability to antagonize Vif binding to A3G. Antagonists are anticipated to be either allosteric inhibitors that prevent Vif and A3G interaction or bind to the interfaces between Vif and A3G and thereby directly inhibit protein-protein interaction. Early on in the critical path additional Aims will triaged hits for dose- dependent protection of A3G from Vif-dependent degradation, specificity as antagonist of the interaction between Vif and A3G and lack of cytotoxicity. Mechanism of action will be confirmed. Antiviral efficacy will be assessed in PBMCs using multiple HIV clades and drug-resistant HIV strains to identify compounds that have a broadly neutralizing potential. Hits that enable the accumulation of A3G-preferred, 'hot spot' sites of dG to dA mutation in viral genomes will be selected. Molecular probes discovered here will enable the field to address open questions concerning the mechanism and efficacy of A3 proteins in restricting HIV replication that otherwise would not be possible in a preclinical environment wherein wild type Vif interacts with natively expressed A3 proteins. They may also provide starting point for future preclinical drug discovery.